Protein iduced by homogeneous blood transfusion and dna encoding the same

ABSTRACT

The present invention relates to a novel protein (MAY-I) which exhibits immunosuppressive activity in allogeneic mixed lymphocyte reaction, and provides a gene encoding that protein, a vector containing said gene, a transformant produced by said vector, a method of manufacturing the said protein with immune activity by culturing said transformant, and a medicinal composition and the like containing the said protein.

TECHNICAL FIELD

The present invention relates to a protein having immunosuppressiveactivity, to a gene encoding the protein, to a vector containing thegene, to a transformant produced by the vector, to a method ofmanufacturing a protein having immunosuppressive activity by culturingthe transformant, to a medicinal composition containing the protein andthe like.

BACKGROUND ART

Organ transplantation is a technique which has already been establishedas a final treatment for end-stage organ failure. From a medicalstandpoint, the greatest problem for organ transplantation i s acute andchronic organ rejection. Clinically effective drugs which are currentlyin use include such powerful immunosuppressants as adrenocorticalhormone, cyclosporin, tacrolimus, azathioprine, anti-thymocyteantibodies and the like. However, these drugs have a general depressanteffect on the host's immune system. Such immunosuppression is usually afactor in the main causes of death following organ transplantation,including organ rejection, infection and malignant tumors. Therefore, itwould seem that in addition to antigen-specific immunosuppression, saferand more successful transplantation could be achieved if immunetolerance was artificially introduced, making it possible for the organto survive permanently with only initial treatment.

Moreover, the aforementioned immunosuppressants are also seen aspromising prophylactic or therapeutic agents for auto-immune disorderssuch as rheumatism and psoriasis and allergic disorders such as allergicasthma (bronchial asthma and the like), allergic rhinitis, allergicconjunctivitis, allergic dermatitis (atopic dermatitis and the like) andpollinosis, or antirejection drug.

It has long been known that renal transplants are more successful inrenal failure patients who receive repeated preoperative bloodtransfusions (Opelz G et al, Lancet 1, 696-698 (1974)). It has also beenrecognized that a more effective immune reaction is induced byallogeneic blood transfusion in which the transfusion is specificallymatched to the donor (donor specific blood transfusion) (Opelz G et al,Transplant Proc 17, 2357-2361 (1985)) and verified by many experimentaltests. In experiments with rodents, complete immune tolerance wasinduced in many cases through a single allogeneic blood transfusion(Marino H et al, Am J Surg 95, 267-273 (1958); Marquet et al, TransplantProc III, 708-710 (1971); Fabre J W et al, Transplantation 14, 608-616(1972)).

There have also been a variety of reports on the mechanism by whichallogeneic blood transfusion might induce immune tolerance. Generallyspeaking, the mechanisms are depending on either cellular or humoralfactors (Kobayashi, Eiji, Molecular Medicine 34, 796-804 (1997)). Clonaldelation (Cranston D et al, Transplantation 42, 302-306 (1986)) andanergy (Dollman M J et al, J Exp Med 173, 79-87 (1991)) belong in theformer mechanism, while the latter mechanism includes bioactivesubstances in vivo induced by allogeneic blood transfusion which arebiologically active in vivo. One of humoral factors is anti-idiotypeantibody, which has been shown to be produced immediately afterallogeneic blood transfusion in animals (NagarkattI P S et al,Transplantation 36, 695-699 (1983); Downey W E III et al,Transplantation 49, 160-166 (1990); Baldwon W M III et al,Transplantation, 51, 481-485 (1991)) and humans (Horuzxko A et al,Immunology Letters 26, 127-130 (1990)), but less is known about theothers.

And since bioactive substances derived through allogeneic transfusionwhich exhibit immunosuppression are endogenous substances, they can beexpected to have fewer side effects than existing immunosuppressants.

DISCLOSURE OF THE INVENTION

Using an allogeneic blood transfusion model to resolve theaforementioned problems, the inventors discovered a novel proteinproduced in the blood which has immunosuppressive activity and perfectedthe present invention.

The present invention provides the following proteins and the like.

Item 1. A protein (a) or (b) below:

-   -   (a) a protein comprising the amino acid sequence shown by SEQ ID        No. 14;    -   (b) a protein comprising the amino acid sequence as defined        in (a) wherein one or more amino acids are deleted, replaced or        added and having immunosuppressive activity.

Item 2. A protein (a) or (b) below:

-   -   (a) a protein having the amino acid sequence shown by SEQ ID No.        1;    -   (b) a protein comprising the amino acid sequence as defined        in (a) wherein one or more amino acids are deleted, replaced or        added and having immunosuppressive activity.

Item 3. A protein (a) or (b) below:

-   -   (a) a protein comprising the amino acid sequence shown by SEQ ID        No. 8;    -   (b) a protein comprising the amino acid sequence as defined        in (a) wherein one or more amino acids are deleted, replaced or        and added and having immunosuppressive activity.

Item 4. A gene encoding the protein as defined in any one of Items 1through 3.

Item 5. A gene comprising a DNA (a) or (b) below:

-   -   (a) a DNA comprising the nucleotide sequence shown by SEQ ID No.        15;    -   (b) a DNA which hybridizes under stringent conditions with a DNA        comprising the nucleotide sequence as defined in (a), and which        encodes a protein having immunosuppressive activity.

Item 6. A gene comprising a DNA (a) or (b) below:

-   -   (a) a DNA comprising the nucleotide sequence shown by SEQ ID No.        2;    -   (b) a DNA which hybridizes under stringent conditions with a DNA        comprising the nucleotide sequence as defined in (a), and which        encodes a protein having immunosuppressive activity.

Item 7. A gene comprising a DNA (a) or (b) below:

-   -   (a) a DNA comprising the nucleotide sequence shown by SEQ ID No.        9;    -   (b) a DNA which hybridizes under stringent conditions with DNA        comprising the nucleotide sequence as defined in (a), and which        encodes a protein having immunosuppressive activity.

Item 8. A protein having an amino acid sequence encoded by the gene asdefined in any one of Items 4 through 7.

Item 9. A vector containing the gene as defined in any one of Items 4through 7.

Item 10. A transformant containing the vector as defined in Item 9.

Item 11. A method of manufacturing a protein comprising:

-   -   Step 1 of culturing the transformant as described in Item 10;        and    -   Step 2 of collecting a protein having immunosuppressive activity        from the culture obtained in the said step.

Item 12. A medicinal composition containing any one of the proteindefined in any one of Items 1 through 3 or Item 8 as an activeingredient, together with a pharmacologically acceptable carrier.

Item 13. The medicinal composition as defined in Item 12, wherein themedicinal composition is an immunosuppressant.

Item 14. The medicinal composition as defined in Item 13, wherein theimmunosuppressant is a prophylactic or therapeutic agent for anauto-immune disorder or allergic disorder, or an antirejection drug.

Item 15. The medicinal composition as defined in Item 14, wherein theauto-immune disorder is rheumatism or psoriasis.

Item 16. The medicinal composition as defined in Item 14, wherein theallergic disorder is bronchial asthma, allergic rhinitis, allergicdermatitis or pollinosis.

Representation of amino acids, peptides, nucleotide sequences, nucleicacids and the like by abbreviations in this description is in conformitywith the rules recommended by the IUPAC-IUB, “Guidelines for WritingDescriptions Containing Nucleotide sequences or Amino Acid Sequences”(edited by Japanese Patent Office), and the conventions relating to useof codes or symbols in the art.

Moreover, in the present invention “gene” (“DNA”) includes not onlydouble-stranded DNA but also single-stranded DNA comprising a sense oranti-sense strand thereof, and there are no limits on its length.Therefore, unless otherwise specified, the gene (DNA) of the presentinvention includes double-stranded DNA including human genome DNA,single-stranded DNA (sense strand) including cDNA, single-stranded DNA(anti-sense strand) having a sequence complementary to the sense strand,and fragments thereof.

The protein having immunosuppressive activity of the present inventioncan be obtained for example by the following method. When transfusing 1ml of the heparinized whole blood of 8-10 week-old DA rats (allogeneicblood transfusion) into the veins or portal veins, preferably the portalveins, of 8-10 week-old Lewis rats, and isolating and purifying from thetissue, cells or blood of the Lewis rats after 4-28 days or preferablyone week, extract obtained by homogenizing the animals' tissue or cellsand then extracting with acid and the like, or preferably whole bloodobtained from the abdominal aorta is collected and centrifuged at 0-20°C., preferably 4° C., and collecting a serum fraction containing theprotein of the present invention.

The protein of the present invention can be purified and isolated fromthe extract or preferably the serum containing the protein of thepresent invention obtained by the aforementioned methods by acombination of such purification methods as salting out, dialysis, gelfiltration, reversed phase chromatography, ion exchange chromatography,affinity chromatography and other forms of chromatography.

The immunosuppressive activity of the protein of the present inventioncan be assayed using a variety of immune reactions employing mouse, rator human lymphocytes, for example immunosuppressive activity can beassayed with high sensitivity by adding the immunosuppressive substancein an allogeneic mixed lymphocyte reaction (MLR) of mice, rats orhumans. A substance which exhibits immunosuppressive activity inallogeneic MLR is promising candidates for immunosuppression. They areparticularly useful as drugs for auto-immune disorders such asrheumatism and psoriasis as well as allergic disorders such as allergicasthma (bronchial asthma and the like), allergic rhinitis, allergicconjunctivitis, allergic dermatitis (atopic dermatitis and the like) andpollinosis, and antirejection drugs.

A 26 kDa protein having immunosuppressive activity (hereinafter referredto below as “MAY-I”) which was newly isolated and identified by thepresent inventors was broken into suitable fragments, and the amino acidsequences of the fragments were determined and compared to known aminoacid sequences. As a result, the presence of fragments having amino acidsequences matching amino acids 699-725, 785-789 or 897-900 of the aminoacid sequence of inter-alpha-inhibitor H4P heavy chain-rat (GeneBankaccessions No. Y11283: hereinafter referred to hereunder as rat IαIH4P)(SEQ ID No. 6) was confirmed. The amino acid sequences matching aminoacids 699-725, 785-789 or 897-900 of rat IαIH4P are given as SEQ ID Nos.3, 4 and 5, respectively.

The expected molecular weight of the protein from amino acid 699 of ratIαIH4P to the C-terminal amino acid was 26 kDa, the same as that ofMAY-I. The inventors then cloned the cDNA sequence (SEQ ID No. 2) ofMAY-I in a polymerase chain reaction (PCR) from the cDNA sequence of ratIαIH4P. This cDNA sequence of MAY-I was then transferred into a proteinexpression vector, preparing recombinant MAY-I. This recombinant MAY-Iexhibited immunosuppressive activity when subjected to allogeneic MLR.Consequently, it was shown that in terms of its structure MAY-I isidentical to a protein having the amino acid sequence of rat IαIH4P fromamino acid 699 to the C-terminal.

In addition, the inventors cloned the entire cDNA sequence (SEQ ID No.9) of rat IαIH4P, prepared a recombinant protein having the amino acidsof SEQ ID No. 8, and performed allogeneic MLR to confirmimmunosuppressive activity. They then removed the sequence (SEQ ID No.2) corresponding to MAY-I from the cDNA sequence of SEQ ID No. 9,prepared a recombinant protein having an amino acid sequence excludingthe amino acid sequence corresponding to MAY-I, and performed allogeneicMLR to confirm that it did not exhibit immunosuppressive activity, thusshowing that it is MAY-I that controls the immunosuppressive function.

Proteins resembling rat IαIH4P also exists in humans, including humanPK-120 and human IHRP, and these proteins as a class are known as IαIH4P(Hitoshi N et al, FEBS Lett 357, 207-211 (1995) (PK-120), Carl H H etal, U.S. Pat. No. 5,459,063 (1989) (sgp120), Ken H et al, J Biochem 119,577-584 (1996) (IHRP)). The amino acid sequence and DNA sequence ofhuman PK-120 are shown by SEQ ID Nos. 12 and 13, respectively, while theamino acid sequence and DNA sequence of human IHRP are shown by SEQ IDNos. 10and 11, respectively. Despite some differences in their aminoacid sequences and DNA sequences, human PK-120 and human IHRP exhibitextremely high homology (homology analysis of GeneBank sequence databaseaccession No. D38595 (human IHRP) and GeneBank sequence databaseaccessions No. D38535 (human PK-120)). Moreover, it is clear that theamino acid sequence of human IHRP (SEQ ID No. 10) and the amino acidsequence of rat IαIH4P (SEQ ID No. 6) are also highly homologous (SoueyE et al, Biochem Biophys Res Commun 243, 522-530 (1998)). The cDNAsequences of the two also exhibit 73% homology (homology analysis of ratIαIH4P nucleotide sequence (SEQ ID No. 7), GeneBank nucleotide sequencedatabase accessions No. Y11283, and human IHRP nucleotide sequence (SEQID No. 11), GeneBank nucleotide sequence accessions No. D38595),suggesting that this protein is preserved across species. We then clonedthe cDNA sequence (SEQ ID No. 15) encoding the amino acid sequence (SEQID No. 14) corresponding to MAY-I in the human IHRP amino acid sequence(SEQ ID No. 10), and combined it with a protein expression vector toprepare recombinant human MAY-I. When subjected to allogeneic MLR, thisrecombinant MAY-I exhibited immunosuppressive activity. This suggeststhat the amino acid sequence (SEQ ID No. 10) of human IHRP whichcontains the amino acid sequence of SEQ ID No. 14, and the amino acidsequence (SEQ ID No. 12) of human PK-120, which is highly homologous tothe amino acid sequence of human IHRP, may have immunosuppressiveactivity in humans similar to that of human MAY-I. It is alsoconceivable that other IαIH4P proteins may also produceimmunosuppression in humans if they contain an amino acid sequenceidentical to that of MAY-I or an amino acid sequence which has beenmodified only to the extent that the immunosuppressive activity of MAY-Iis not lost.

Since the nucleotide sequence of pig IαIH4P (SEQ ID No. 18) (Ken H etal, J Biochem 119, 577-584 (1996)) is also highly homologous with theDNA sequence of rat IαIH4P, it is likely that as in the case of humanIαIH4P, a protein comprising the amino acid sequence of pig IαIH4P (SEQID No. 17) or the amino acid sequence (SEQ ID No. 19) corresponding toMAY-I therein would have immunosuppressive activity in humans. Anucleotide sequence encoding for an amino acid sequence corresponding tothe MAY-I segment of the amino acid sequence of pig IαIH4P is shown asSEQ ID No. 20.

Moreover, an amino acid sequence corresponding to MAY-I in the aminoacid sequences of the IαIH4P of other mammals might also have the sameimmunosuppressive activity as pig IαIH4P. The present inventionencompasses (a) a protein comprising an amino acid sequencecorresponding to MAY-I in the amino acid sequences of the IαIH4P ofmammals other than rats, humans and pigs, or (b) a protein comprising anamino acid sequence (a) with one or more amino acids deleted, replacedor added and having immunosuppressive activity. In addition, the presentinvention encompasses gene encoding aforementioned amino acid sequencesin (a) or (b).

The protein of the present invention has an immunosuppressive effect.The amino acid sequence expressed by SEQ ID No. 14 is an amino acidsequence which was induced based on the nucleotide sequence of a genewhich was cloned in an Example of this Description. The protein of thepresent invention is a protein comprising the amino acid sequenceexpressed by SEQ ID No. 14. The present invention also encompasses aprotein comprising this amino acid sequence with one or more amino acidsdeleted, replaced or added and having immunosuppressive activity. In thepresent Description, “more” in “one or more” signifies 2-50 orpreferably 2-30 or more preferably 2-20 or ideally 2 to a few.

Moreover, a protein of the present invention is a protein comprising theamino acid sequence expressed by SEQ ID No. 1. The present inventionalso encompasses a protein comprising this amino acid sequence with oneor more amino acids deleted, replaced or added and havingimmunosuppressive activity.

Moreover, a protein of the present invention is a protein comprising theamino acid sequence expressed by SEQ ID No. 8. The present inventionalso encompasses a protein comprising this amino acid sequence with oneor more amino acids deleted, replaced or added and havingimmunosuppressive activity.

The present invention also encompasses a protein havingimmunosuppressive activity which comprises amino acids having 70% orgreater, preferably 80% or greater or more preferably 95% or greaterhomology with the amino acid sequences of any of SEQ ID Nos. 14, 1 or 8.Moreover, a gene encoding said proteins is also included in the presentinvention.

In general, naturally-occurring proteins may be subject to deletion,addition, replacement and other changes to radicals in the amino acidsequence due to polymorphisms or mutations in the gene encoding thereforor to modifications after protein formation, but nonetheless retain thesame physiological activity as the unmutated protein. It is alsopossible to artificially create genetic mutations using the techniquesof gene recombination, in such a way that the physiological activity ofthe protein is effectively unchanged. A protein comprising the aminoacid sequence of SEQ ID Nos. 1, 8 or 14 which has been altered by such anatural or artificial mutation is also included in the proteins of thepresent invention as long as it retains the immunosuppressive function,and naturally or artificially mutated gene is included in the gene ofthe present invention as long as the protein comprising the amino acidsequence encoded by said gene retains its immunosuppressive action.Alleles of these are also included.

Methods of creating the artificial mutations include genetic engineeringtechniques such as site specific mutagenesis (Methods in Enzymology154:350 & 367-382, 1987 and 100:468, 1983; Nucleic Acids Res 12:9441,1984; A Course in Successive Chemical Experimentation I, “GeneticResearch II”, Nihon Seikagakukai p105, 1986), techniques of chemicalsynthesis such as the phosphotriester and phosphoamidite methods (J AmChem Soc 89:4801, 1967 and 91:3350, 1969; Science 150:178, 1968;Tetrahedron Lett 22:1859, 1981 and 24:245, 1983), and combinations ofthese methods. Specifically, DNA synthesis may be by chemical synthesisusing the phosphoamidite method or phosphotriester method, and may beperformed on a commercially available automated oligonucleotidesynthesizer. Double-strand fragments may be obtained from the chemicallysynthesized single-strand product by either synthesizing a complementarystrand and annealing the strands together under appropriate conditions,or by adding the complementary strand using DNA polymerase together withan appropriate primer sequence.

There are no particular limitations on the origin of the protein of thepresent invention, which may be a natural protein, a recombinant proteinor a chemically synthesized protein. When a natural protein is desired,a culture of tissue or cultured cells expressing the target protein maybe used as the starting material, and purification accomplished by asuitable combination of well-known methods of protein purification suchas salting out, affinity chromatography, ion exchange chromatography,gel filtration and the like. For example, when affinity chromatographyis used the target protein can be purified using a carrier to which havebeen bound antibodies against the protein of the present invention.

When a recombinant protein is desired, a recombinant expression vectorobtained by cloning DNA of the present invention which encodes theaforementioned target protein in a suitable expression vector istransferred to a host (E. coli, yeast etc.), and the transformantcultured under suitable conditions to produce the target protein. Forpurposes of isolating the target protein, it is generally desirable thatthe target protein by secreted into the culture supernatant, which canbe achieved by optionally selecting the combination of recombinationvector and host and culture conditions. Manufacture of a proteincomprising the desired amino acid sequence by chemical synthesis canalso be done optionally by the person skilled in the art.

Suitable pharmacologically acceptable modifications can also be added tothe protein of the present invention as long as its immunosuppressiveactivity is maintained. That is, although the proteins comprising theamino acid sequences shown by SEQ ID Nos. 14, 1 and 8 or comprising apartial amino acid sequence shown thereby normally have a carboxyl(—COOH) or carboxylate (—COO—) group at the C-terminal, the C-terminalmay also be an amide (—CONH₂) or ester (—COOR). The R of the ester maybe for example C₁₋₆ alkyl group such as a methyl, ethyl, n-propyl,isopropyl, n-butyl, C₃₋₈ cycloalkyl group such as a cyclopentyl,cyclohexyl, C₆₋₁₂ aryl group such as a phenyl, α-naphthyl, C₇₋₁₄ aralkylgroup such as phenyl-C₁₋₂ alkyl (e.g., a benzyl, phenethyl, benzhydryland the like), α-naphthyl-C₁₋₂ alkyl (e.g., an α-naphthylmethyl and thelike), or pivaloyloxymethyl ester, which is a widely used ester for oraluse. Possible salts of the protein of the present invention includepharmacologically acceptable bases (such as alkali metals) and acidsalts (organic and inorganic acids), pharmacologically acceptableacid-added salts are particularly desirable. Examples of such saltsinclude salts of inorganic acids (such as hydrochloric acid, phosphoricacid, hydrobromic acid and sulfuric acid) and salts of organic acids(such as acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). Theprotein of the present invention and precursors, amides and estersthereof have immunosuppressive activity and are useful as drugs and inparticular as prophylactic and therapeutic agent for auto-immunedisorders (rheumatism, psoriasis and the like) or allergic disorders(allergic asthma (bronchial asthma), allergic rhinitis, allergicconjunctivitis, allergic dermatitis (atopic dermatitis), pollinosis andthe like), and antirejection drug.

The gene of the present invention encodes a protein havingimmunosuppressive activity. Specifically, the gene of the presentinvention is DNA which encodes a protein having the amino acid sequenceof either SEQ ID No. 14, 1 or 8, or one of these with one or more aminoacids deleted, replaced or added, and having immunosuppressive activity.Moreover, the gene of the present invention contains a DNA whichcomprises the nucleotide sequence of either SEQ ID No. 15, 2 or 9, orwhich hybridizes under stringent conditions with DNA with such anucleotide sequence, and encodes a protein having immunosuppressiveactivity. The gene of the present invention can also be used in genetherapy.

There are no particular limits on the stringent hybridizationconditions, although in general, conditions are selected so that theprobe DNA sequence and the DNA sequence to be detected are as homologousas possible. Stringent hybridization conditions can be achieved byadjusting the solvent concentration and/or salt concentration of thehybridization solution, the hybridization temperature, the hybridizationtime and the like. The washing conditions after hybridization (saltconcentration of the washing liquid and the like) can also be adjusted.Such conditions can be suitably selected by the person skilled in theart depending on the length and/or base composition of the probe, andthe degree of homology between the nucleotide sequence to be detectedand the nucleotide sequence of the probe.

The gene of the present invention can also be manufactured by thefollowing genetic engineering methods. Methods of cloning the gene ofthe present invention including using a synthetic DNA primer having apartial nucleotide sequence of the protein of the present invention toamplify the target DNA from genome DNA, genome DNA library or thetissues, cells or preferably liver of humans or warm-blooded animals byknown PCR methods, or selecting DNA incorporated into a suitable vectorby hybridization with labeled DNA with synthetic DNA or DNA fragmentshaving a part or all of the regions of the protein of the presentinvention. Methods of hybridization include for example those describedin Molecular Cloning (2nd Ed., J Sambrook et al, Cold Spring Harbor LabPress, 1989). When using a commercial library, the methods described inthe attached manual may be employed. The cloned DNA encoding the proteinof the present invention can be used either as is or if desired may bedigested with a restriction enzyme or have a linker added. Said DNA mayhave ATG as the translation start codon at the 5′ terminal, or TAA, TGAor TAG as the translation stop codon at the 3′ terminal. These start andstop codons may also be added using a suitable synthetic DNA adapter.

A vector containing the gene of the present invention is provided by thepresent invention. There are no particular limits on the type of vector,which can be selected according to the purpose for which it will beused. In general it is possible to use plasmid vectors and phagevectors, which are available commercially. An expression vector is usedin order to produce the recombinant proteins encoded by the gene of thepresent invention.

The expression vector for the protein of the present invention can bemanufactured for example by (a) cutting the target DNA fragment from theDNA encoding for the protein of the present invention, and (b) attachingsaid DNA fragment downstream the promoter in a suitable expressionvector. Vectors which may be used include plasmids derived from E. coli(such as pBR322, pBR325, pUC12 and pUC13), plasmids derived fromBacillus subtilis (such as pUB110, pTP5 and pC194), plasmids derivedfrom yeasts (such as pSH19 and pSH15), bacteriophages such as λ phage,retroviruses, Vaccinia virus, Baculoviridae and other animal viruses.The promoter used in the present invention may be any promoter suited tothe host used to express the DNA.

A transformant produced by transferring a recombinant vector into a hostis provided by the present invention. Any suitable living creature canbe used as the host, such as for example eucaryotic microorganisms(animals cells, plant cells, yeasts and the like) and prokaryoticmicroorganisms (E. coli and the like). Methods known by the personskilled in the art can be used for transformation, includingspecifically the calcium phosphate, electroporation, microinjection andlipofection methods and the like.

When the host for transformation consists of animal cells, aSV40-derived promoter, retrovirus promoter, metallothionein promoter,heat shock promoter, cytomegalovirus promoter or SRα promoter or thelike may be used. When the host is an Escherichia, a trp promoter, T7promoter, lac promoter, recA promoter, λPL promoter, 1 pp promoter orthe like is desirable, while if it is a Bacillus, an SPO1 promoter, SPO2promoter, penP promoter or the like is desirable, and if the host is ayeast, a PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter or GALpromoter or the like is preferred. When the host consists of insectcells, a polyhedrin promoter or P10 promoter or the like is desirable.In addition, the expression vector may contain enhancers, splicingsignals, PolyA addition signals, selection markers and SV40 replicationorigins (sometimes abbreviated herein as SV40ori). Possible selectionmarkers include for example the dihydrofolic acid reductase (sometimesabbreviated herein as dhfr) gene (methotrexate (MTX) resistant),ampicillin resistance gene (sometimes abbreviated herein as Ampr) andneomycin resistance gene (G418 resistant, sometimes abbreviated hereinas Neo). In particular, when using CHO (dhfr⁻) cells and the DHFR geneas the selection marker, selection can also be accomplished with amedium that does not contain thymidine. If necessary, a signal sequencematched to the host can also be added to the N-terminal of the proteinor partial peptide thereof. A phoA signal sequence, OmpA signal sequenceor the like can be used if the host is an Escherichia, an α-amylasesignal sequence, subtilisin signal sequence or the like if the host is aBacillus, a mating factor α (MFα) signal sequence, invertase signalsequence or the like if the host is a yeast, and an insulin signalsequence, α-interferon signal sequence, antibody molecule signalsequence or the like if the host is animals cells. The transformant canbe manufactured using a vector containing DNA constructed as noted aboveencoding for the protein.

Possible hosts include for example Escherichia, Bacillus, yeasts,insects, insect cells, animal cells and the like. Of the Escherichia,Escherichia coli K12 DH1 (Proc Natl Acad Sci USA 60, 160, 1968), JM103(Nucleic Acids Research 9, 309, 1981), JA221 (Journal of MolecularBiology 120, 517, 1978), HB101 (Journal of Molecular Biology 41, 459,1969), C600 (Genetics 39, 440, 1954) or the like can be used. Of theBacillus, Bacillus subtilis MI114 (Gene 24, 255, 1983) or.207-21(Journal of Biochemistry 95, 87, 1984) or the like can be used.

Yeasts such as Saccaromyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5Dand 20B-12 can be used. Possible insects include for example bombiclarvae (Maeda et al., Nature, Vol. 315, 592 (1985)). In terms of insectcells, for example if the virus is AcNPV, established cell lines derivedfrom Spodoptera frugiperda larvae (Sf cells), MG1 cells from themid-intestines of Trichoplusia ni, High Five TM cells from Trichoplusiani eggs, or cells derived from Mamestra brassicae, Estigmena acrea orthe like may be used. If the virus is BmNPV, an established Bombyx moriN cell line (BmN cells) or the like may be used. Sf cells that may beused include for example Sf9 cells (ATCC CRL1711) and Sf21 cells (bothfrom Vaughn, J L, In Vitro 13, 213-217, 1977). Animal cells which may beused include for example monkey COS-7 cells, Vero cells, CHO chinesehamster cells, CHO chinese hamster cells lacking the DHFR gene (dhfr-CHOcells), mouse L cells, mouse 3T3 cells, mouse myeloma cells, humanHEK293 cells, human FL cells, 293 cells, C127 cells, BALB 3T3 cells,Sp-2/O cells and the like. Transformation of Escherichia can beaccomplished for example by the methods described in Proc Natl Acad SciUSA 69,. 2110, 1972 or Gene 17, 107, 1982 or the like. Transformation ofBacillus can be accomplished for example by the methods described inMolecular & General Genetics 168, 111, 1979. Transformation of yeastscan be accomplished for example by the methods described in Proc NatlAcad Sci USA 75, 1929, 1978.

Transformation of insect cells or insects can be accomplished-forexample by the methods described in Bio/Technology 6, 47-55, 1988.Transformation of animal cells can be accomplished for example by themethods described in Virology 52, 456, 1973. Introduction of theexpression vector into the cells can be accomplished for example by thelipofection method (Felgner, P L et al, Proceedings of the NationalAcademy of Sciences of the United States of America 84, 7413, 1987), thecalcium phosphate method (Graham, F L and van der Eb, A J, Virology 52,456-467, 1973) or the electroporation method (Neumann E et al, EMBO J.1, 841-845, 1982) or the like. A transformant transformed by anexpression vector containing DNA encoding the protein of the presentinvention is obtained in this way. Methods of stably expressing theprotein, etc. of the present invention using animal cells includemethods of selecting by clone selection those cells in which theexpression vector introduced into the cells has been incorporated intothe chromosomes. Specifically, transformants are selected using theaforementioned selection marker as the reference. Moreover, a stableanimal cell strain with high expression of the protein, etc. of thepresent invention can be obtained by repeated clone selection of animalcells obtained in this way using a selection marker. When the dhfr geneis used as the selection marker, DNA encoding the protein, partialpeptide thereof or the like of the present invention can be amplified inthe cells together with the dhfr gene by gradually increasing the MTXconcentration of the culture and selecting for resistance strain, toobtain an animal cell strain with even higher expression. The protein orthe like of the present invention can then be manufactured by culturingthe transformant in conditions under which DNA encoding the protein orthe like of the present invention can be expressed, and producing andaccumulating the protein or the like of the present invention.

There are no particular limitations on the medicinal composition of thepresent invention as long as it contains the protein of the presentinvention. The medicinal composition of the present invention may alsocontain physiologically allowable carriers, excipients and the like asusage in addition to the protein of the present invention.

The immunosuppressant of the present invention is useful as aprophylactic or therapy for auto-immune disorders (rheumatism, psoriasisand the like) or allergic disorders (allergic asthma (bronchial asthma),allergic rhinitis, allergic conjunctivitis, allergic dermatitis (atopicdermatitis), pollinosis and the like), and antirejection drugs.

Conventional methods can be employed when the protein or DNA encodingtherefor of the present invention is used as the aforementionedmedicinal composition. For example, if necessary it may be administeredorally in the form of a sugar-coated or enteric-coated tablet, capsule,elixir, microcapsules or the like, externally as an ointment, plaster orthe like, nasally as a spray, inhalant or the like, or parenterally byinjecting a suspension or sterile solution made with water or otherpharmacologically acceptable liquid. Possible methods of administeringthe protein of the present invention for treatment of organ transplantrejection include oral administration, injection, intraarticularadministration, intrarectal administration, perfusion for thetransplanted organ, administration to the transplanted organ andadministration through a balloon catheter. It can be manufactured forexample by formulating the compound or salt thereof together withphysiologically acceptable carriers, flavorings, excipients, vehicles,preservatives, stabilizers, binders and the like in the dosage formrequired by generally accepted pharmaceutical practice. The amount ofactive component in such formulations is designed to provide a dosewithin the indicated range. The best modes of administration areinjection, inhalation, nasal drops, external administration and otherforms of topical administration.

Additives which may be blended into tablets and capsules include forexample binders such as gelatin, corn starch, gum tragacanth and gumarabic, excipients such as crystal cellulose, swellings such as cornstarch, gelatin and alginic acid, lubricants such as magnesium stearate,sweeteners such as sucrose, lactose or saccharin, and flavorings such aspeppermint, akamono oil and cherry. A capsule formulation may alsocontain oils and other liquid carriers in addition to the previous typesof ingredients. Sterile compositions for purposes of injection may beformulated by ordinary methods such as dissolving or suspending activeingredients, sesame oil, coconut oil and other naturally vegetable oilsin a vehicle such as injectable water. Aqueous injections include forexample physiological saline and isotonic solutions containing glucoseand other adjuvants (such as D-sorbitol, D-mannitol, sodium chloride andthe like), and suitable solubilizers such as alcohols (i.e. ethanol),polyalcohols (i.e. propylene glycol, polyethylene glycol) and nonionicsurfactants (i.e. polysorbate 80 (TM), HCO-50) may also be added. Oilyliquids such as sesame oil and soy bean oil may also be added, as cansolubilizers such as benzyl benzoate and benzyl alcohol. Buffers (suchas phosphate buffers and sodium acetate buffers), analgesics (such asbenzalconium chloride and procaine hydrochloride), stabilizers (such ashuman serum albumin and polyethylene glycol), preservatives (such asbenzyl alcohol and phenol) and antioxidants may also be used. Theprepared injection is normally used to fill ampoles. Since the resultingformulation is stable and of low toxicity, it can be administered forexample to humans and other mammals (such as mice, rats, guinea pigs,rabbits, chickens, sheep, pigs, cows, cats, dogs, monkeys, sacredbaboons, chimpanzees and the like).

The dosage per day of the protein of the present invention or DNAencoding therefor varies depending on symptoms and the like, but in thecase of oral administration it is normally between about 0.0001 g and 10g or preferably about 0.1 mg and 100 mg or more preferably about 1.0 mgand 50 mg or ideally about 1.0 mg and 20 mg per day for an adulttransplant patient (weight 60 kg). In the case of parenteraladministration, the single dosage varies depending on the patient,organ, symptoms and method of administration. For example when theprotein of the present invention or DNA coding therefor is injected toan adult transplant patient (weight 60 kg), an intravenous injection ata dosage between about 0.00001 g and 1 g or preferably about 0.01 mg and30 mg or more preferably about 0.1 mg and 20 mg or ideally about 0.1 mgand 10 mg per day is desirable. The dosage is applicable to otheranimals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the immunosuppressive activity of allogeneic transfusionserum in allogeneic rat MLR;

FIG. 2 shows a chromatogram of allogeneic transfusion serum by protein Gaffinity column;

FIG. 3 shows the immunosuppressive activity in allogeneic rat MLR of thefraction that did not bind to the protein G affinity column;

FIG. 4 shows a hydroxyapatite chromatograph of the fraction that did notbind to the protein G affinity column;

FIG. 5 shows the immunosuppressive activity in allogeneic rat MLR of thefraction isolated by hydroxyapatite chromatography;

FIG. 6 shows a gel filtration chromatogram of the fraction recognized byhydroxyapatite chromatography as having immunosuppressive activity;

FIG. 7 shows the immunosuppressive activity in allogeneic rat MLR of thefraction isolated by gel filtration chromatography;

FIG. 8 is an SDS-polyacrylamide gel electrophoresis photograph of theFr. 28 isolated by gel filtration chromatography, showing that it is asingle protein;

FIG. 9 shows the immunosuppressive activity in allogeneic rat MLR ofrecombinant rat MAY-I;

FIG. 10 shows the immunosuppressive activity in allogeneic rat MLR ofrecombinant rat MAY-I, IαIH4P and partial-H4P; and

FIG. 11 shows the immunosuppressive activity in allogeneic human MLR ofrecombinant human MAY-I.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained in more detail below with referenceto examples, but is not limited by these examples.

EXAMPLE 1 Immunosuppressive Activity Induced by Allogeneic BloodTransfusion

1 ml of the heparinized whole blood of 8-10 week-old DA rats wastransfused into the portal veins of 8-10 week-old Lewis rats (allogeneictransfusion), and one week later all blood was collected from theabdominal aortas of the Lewis rats. As a control, 1 ml of theheparinized whole blood of Lewis rats was also transfused into theportal veins of Lewis rats (blood transfusion: abbreviated hereunder as“BT”), and one week later all blood was collected from the abdominalaortas of the Lewis rats. The blood of the subject rats and that ofcontrol rats was centrifuged at 4° C., and the serum fraction collected(referred to hereunder as allogeneic transfusion serum and BT serumrespectively). Protein concentrations in the allogeneic transfusion andBT serum were measured, concentrations of 1, 0.4 and 0.1 μg were addedto allogeneic rat MLR, and the immunosuppressive activity was compared.Protein concentrations in the serums were measured using a commercialBCA protein assay kit (Pierce) in accordance with the encloseddirections. In the allogeneic MLR, Lewis rat spleen cells were used asthe reaction cells and DA rat mitomycin C-treated (or irradiated) spleencells as the stimulus cells, and the two cultured together in equalamounts.

The reaction cells were prepared as follows. Spleens were removed from8-10 week-old Lewis rats, and lymphocytes were prepared by specificgravity centrifugation using Lympholyte®-Rat (Cedarlane). Thelymphocytes were adjusted to 10⁶/ml using an RPMI-1640 medium (NikkenBiomedical Laboratory, containing penicillin 100 units/ml, streptomycin100 μg/ml, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate 10 mM,2-mercaptoethanol 55 μM) supplemented with 10% heat inactivated fetalbovine serum (hereunder “FBS”), and used as the reaction cell suspensionwherein the reaction cells floated. The stimulus cells were prepared asfollows. Spleen cells were removed from 8-10 week-old DA rats, andlymphocytes prepared by specific gravity centrifugation usingLympholyte®-Rat. The lymphocytes were suspended in RPMI-1640 mediumsupplemented with 10% FBS, and treated with 25 μg/ml mytomycin C at 37°C. for 15 minutes. After washed three times, they were adjusted to10⁶/ml using RPMI-1640 containing 10% FBS, and used as the stimulus cellsuspension wherein the stimulus cells floated. 100 μl of the reactioncell suspension and 100 μl of the stimulus cell suspension prepared asdescribed above, together with 2 μl of the specimen, were added to96-hole U-bottom microtest plate, and cultured for 3 days at 37° C.under the condition of 5% carbon dioxide and 95% air. Blastogenesis oflymphocytes in allogeneic rat MLR was measured using ³H-thymidineincorporation as the marker. That is, 1 μuCi/well of ³H-thymidine wasadded 18 hours before completion of the culture, and after completion ofculture cells were collected in a cell harvester, and radioactivity inthe cells was measured with a microplate scintillation counter and usedas a marker of allogeneic MLR lymphocyte blastogenesis. The suppressiveactivity of the allogeneic rat MLR was evaluated by calculating thesuppression rate according to the formula below.Suppression rate (%)={1−(cpm of MLR with specimen added−cpm of reactioncells only)/(cpm of MLR without specimen−cpm of reaction cellsonly)}×100.

The results show that allogeneic transfusion serum exhibits obviousimmunosuppressive activity at a protein mass of 0.4 μg (FIG. 1).

EXAMPLE 2 Isolation and Purification of a Bioactive Substance (Protein)which Suppresses Allogeneic Rat MLR

The allogeneic transfusion serum obtained in Example 1 was isolated andpurified by the following methods.

1. Salting Out

The allogeneic transfusion serum was salted out with 40% ammoniumsulfate and the precipitate dissolved in 20 mM sodium phosphate buffer(pH 7.0), and the solution was dialyzed. This solution was centrifugedat 4° C., and the supernatant collected.

2. Protein G Affinity Column Chromatography

The salted-out sample was isolated and purified by medium-pressurechromatography using a Protein G column (Pharmacia) (FIG. 2). Isolationwas performed with a binding solution of 50 mM potassium phosphatebuffer (pH 7.0), an eluant of 100 mM glycine-hydrochloric acid solution(pH 2.7) and a flow rate of 200 μl/minute. The resulting proteins No.2-7 (described hereunder as “Protein G flow through” or “PGFT”)including proteins that did not bind to the column exhibited clearimmunosuppressive activity in allogeneic rat MLR (FIG. 3).

3. Hydroxyapatite Chromatography

The PGFT was isolated and purified by medium-pressure chromatographyusing a CHT20 (Bio-Rad) column (FIG. 4). Isolation was performed with abinding solution of 50 mM potassium phosphate buffer (pH 6.8), an eluantof 500 mM potassium phosphate buffer (pH 6.8), a gradient capacity of300 ml and a flow rate of 2 ml/minute. When the immunosuppressiveactivity of the resulting isolated fractions was investigated byallogeneic rat MLR, Fractions No. 39-42 exhibited immunosuppressiveactivity (referred to hereunder as “CHT 39-42”, FIG. 5).

4. Gel Filtration Chromatography

CHT 39-42 were isolated and purified by medium-pressure chromatographyusing a HiLoad Superdex 200 pg (Pharmacia) (FIG. 6). Isolation wasperformed with 20 mM sodium phosphate buffer/150 mM sodium chloride (pH7.4), at a flow rate of 0.8 ml/minute. When the immunosuppressiveactivity of the isolated fractions was investigated by allogeneic ratMLR, Fractions No. 28 and 29 (described below as “SD28” and “SD29”)exhibited immunosuppressive activity (FIG. 7).

EXAMPLE 3 Confirmation by SDS-PAGE of Purity of Bioactive Substance(Protein) which Suppresses Allogeneic Rat MLR

The purity of the protein in the purification process was evaluated bySDS-polacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performedaccording to the methods described in Protein Test Notes (Part2)—Through Determination of Primary Structure (Yodosha), pp.14-19, andfollowing electrophoresis the gel was stained according to the silverstain method described on p. 22 of the above. The results of SDS-PAGEshowed that SD 28 and 29 were one type of protein (protein contained inSD28 is referred to below as MAY-I, FIG. 8).

The purity of the MAY-I having immunosuppressive activity obtained bythese purification and isolation methods reached about 1.5×10⁸ times(Table 1). TABLE 1 Purification stage Content (ml) Protein volume (mg)Purity Serum 182 14924000 1.0 Salting out 80 816000 18.3 Protein G 78476580 31.3 CHT20 20 15120 987.0 Superdex 200 pg 20 0.098 152909836.1

EXAMPLE 4 Analysis of N-Terminal and Internal Partial Amino AcidSequence of a Protein (MAY-I) which Suppresses Allogeneic Rat MLR

The amino acid sequence of the protein (MAY-I) purified in Example 2which is contained in SD28 and 29 and has immunosuppressive activity wasdetermined. The N-terminal amino acid sequence of the protein wasanalyzed by a protein sequencer (G1005A Protein Sequencing System,Hewlett-Packard) according to the phenyl isothiocyanate method. Theinternal partial amino acid sequence of the protein was analyzed bybreaking the protein's disulfide bonds by carboxymethylization,fragmenting it with lysyl endopeptidase, isolating the peptides withreverse-phase HPLC and analyzing them with the aforementioned proteinsequencer. The same amino acid sequence (27 amino acids from theN-terminal, SEQ ID No. 3) was obtained from both fractions. The aminoacid sequences shown by SEQ ID Nos. 4 and 5 were also found inside theprotein.

EXAMPLE 5 Analysis of Molecular Weight of Bioactive Substance (MAY-I)which Suppresses Allogeneic Rat MLR

The molecular weight of MAY-I was measured by ion spray mass analysisusing a mass spectrometer (API3000, Perkin Elmer Sciex). The resultsshow a protein with a molecular weight of 26089.84 Da.

EXAMPLE 6 Identification of a Gene Fragment Encoding Rat IαIH4P

The 27 N-terminal amino acids (SEQ ID No. 3) analyzed in Example 4 weresubjected to a homology search on the protein database recorded on theGenomeNet FASTA Server (Kyoto Center). The results showed perfecthomology with amino acids number 699-725 of the amino acid sequence ofrat IαIH4P, recorded as accession number JC5953. The amino acidsequences shown as SEQ ID Nos. 4 and 5 were shown to be completelyhomologous with amino acids 785-789 and 897-900, respectively, of theamino acid sequence of rat IαIH4P.

Moreover, the anticipated molecular weight of the protein from aminoacid 699 to the C-terminal amino acid of rat IαIH4P, which was shown tobe homologous with the 27 amino acids of SEQ ID No. 3, was calculated tobe 26080.07 Da, or effectively identical to the molecular weight of theprotein (MAY-I) shown in Example 5. Consequently, this protein encodesthe sequence beginning with amino acid 699 of rat IαIH4P.

EXAMPLE 7 Cloning of DNA Encoding the Sequence (MAY-I) Beginning withAmino Acid 699 of the Amino Acid Sequence of the Rat IαIH4P Obtained inExample 6

The part corresponding to the amino acids from 699 through theC-terminal was cloned by PCR from the total cDNA sequence of rat IαIH4Pas recorded in the GeneBank database under accessions number Y11283.Namely, 1 ml of the heparinized whole blood of 9 week-old DA rats wastranfused (allogeneic transfusion) into the portal veins of 9 week-oldLewis rats, the livers of which were removed one week later and used toprepare total RNA using Isogen (Nippon Gene) according to the manual.cDNA was synthesized with M-MLV reverse transcriptase (GIBCO BRL) fromthe resulting 10 μg of total RNA using a 6-base random primer (TAKARA).PCR was performed with the resulting 1 μg of cDNA as the template. Thenucleotide sequences shown by SEQ ID Nos. 21 and 22 were prepared as thePCR primers. The PCR reaction was performed with an Advantage 2 PCR Kit(Clontech) using 2 μl of each primer, 1 μl of AdvanTaq DNA polymerase,the reaction buffer included with the enzymes, dNTPs and 1 μl of thecDNA, with a total capacity of 40 μl. After the template DNA wasthoroughly denatured through 1 minute of heat treatment at 94° C., acycle of 1 minute at 94° C., 1 minute at 62° C. and 1 minute at 68° C.was repeated 25 times, followed by the elongation reaction for 3 minutesat 68° C. After completion of the reaction, 1.2% agarose gelelectrophoresis was performed using 10 μl of the reaction liquid and theamplification product detected with an ethidium bromide stain. A roughly700 bp band was then removed with a razor blade, centrifugally filtered(UltraFree, Millipore), phenol extracted and ethanol precipitated, and aDNA fragment collected. This DNA fragment was digested with restrictionenzymes BamH I and Xho I, subcloned to the BamH I and Xho I sites ofprotein expression vector psec Tag2 B (Invitrogen), and introduced intoE. coli DH5α to obtain E. coli DH5α/MAY I-pSec Tag2 B. A sequencingreaction was performed on the nucleotide sequence of the cDNA fragmentinserted into the resulting transformant with an ABI PRISM DyeTerminatorCycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems)sequencing reaction, using a T7 primer and a pcDNA 3.1/BGH reverseprimer. Analysis of this cDNA sequence with an ABI PRISM 377 DNASequencer produced a cDNA sequence (SEQ ID No. 2) encoding for theprotein (MAY-1) between amino acid 699 of rat IαIH4P and the C-terminalamino acid.

EXAMPLE 8 Cloning the Total cDNA Nucleotide Sequence of Rat IαIH4P

PCR was performed using 1 μg of the rat liver cDNA synthesized inExample 7 as the template. The nucleotide sequences shown by SEQ ID Nos.23, 24, 25, 26, 27 and 28 were synthesized as the PCR primers. The PCRreaction was performed by the same methods as in Example 7. Followingagarose gel electrophoresis, the DNA fragment (H4P-1) amplified by SEQID Nos. 23 and 24, the DNA fragment amplified by SEQ ID Nos. 25 and 26(H4P-2) and the DNA fragment amplified by SEQ ID Nos. 27 and 28 (H4P-3)were collected from the gel, digested with restriction enzymes BamH Iand EcoR I, subcloned to the BamH I and EcoR I sites of proteinexpression vector pEF4/Myc-His C (Invitrogen), and introduced into E.coli SCS110 (Stratagene) to obtain E. coli SCS110/H4P-1, 2 & 3. Afterconfirmation of the nucleotide sequence of the inserted cDNA, theplasmid vectors containing fragments H4P-1 and H4P-2 were digested withrestriction enzymes BamH I, EcoR I and Bgl II, subcloned again intopEF4/Myc-His C and introduced into E. coli SCS110 to obtain E. coliSCS110/partial-H4P which did not include cDNA encoding for the MAY-Iprotein. After confirmation of the nucleotide sequence of the resultingpartial-H4P cDNA, it was digested together with H4P-3 using restrictionenzymes BamH I, EcoR I and Xba I, subcloned again into pEF4/Myc-His C,and introduced into E. coli DH5α to obtain E. coli DH5α/H4P containingthe total cDNA nucleotide sequence of rat IαIH4P.

EXAMPLE 9 Preparation of the Protein (MAY-I) Corresponding to the AminoAcid Sequence Between 699 and the C-Terminal Amino Acid of the RatIαIH4P Obtained in Example 7, and Preparation of the Complete Rat IαIH4PProtein (H4P) and the Protein (partial-H4P) Excluding the cDNA Encodingthe Amino Acid Sequence of MAY-I Obtained in Example 8

The MAY-I-pSec Tag2 B prepared in Example 7 and the H4P and partial-H4Pprepared in Example 8 were transfectioned to COS 7 cells by theDEAE-Dextran method. The day after transfection, they were transferredto an FBS-free medium and cultured for 24 hours, after which the culturesupernatant was collected and the recombinant MAY-I, H4P and partial-H4Ptherein isolated and purified by the following methods. Namely, theculture supernatant was isolated and purified by medium-pressurechromatography using an His-Trap column (Amersham-Pharmacia). Isolationwas performed with a 20 mM sodium phosphate buffer-10 mM imidazolesolution (pH 7.4) as the binding solution and a 20 mM sodium phosphatebuffer-500 mM imidazole solution (pH 7.4) as the eluant, at a flow rateof 1.5 ml/minute. The buffer of this chromatography peak was exchangedby medium-pressure chromatography using a Hi Trap Desalting column. Theconditions were 10 mM sodium phosphate buffer-120 mM sodium chloridebuffer (pH 7.4), flow rate 1 ml/minute.

EXAMPLE 10 Immunosuppressive Activity of the Recombinant MAY-I, H4P andPartial-H4P Prepared in Example 9

The immunosuppressive activity in allogeneic rat MLR of the recombinantMAY-I (SEQ ID No. 1) prepared in Example 9 was investigated as inExample 1, and concentration-dependent immunosuppressive activity wasshown (FIG. 9). When the immunosuppressive activity of the recombinantH4P (SEQ ID No. 8) and partial H4P was investigated in the same way, theformer exhibited immunosuppressive activity but the latter did not (FIG.10). These results confirm that MAY-I controls immunosuppressiveactivity.

EXAMPLE 11 Cloning of DNA Encoding Human MAY-I (1)

Total RNA was prepared from Hep G2 cells using Isogen (Nippon Gene)according to the manual. Using a 6-base random primer (TAKARA), cDNA wassynthesized from the resulting 10 μg of total RNA with M-MLV reversetranscriptase (GIBCO BRL). PCR was performed using 1 μg of thesynthesized cDNA as a template. The nucleotide sequences shown by SEQ IDNos. 29 and 30 were synthesized as the PCR primers. For the PCRreaction, the template DNA was first thoroughly denatured by 1 minute ofheat treatment at 95° C., then a cycle of 30 seconds at 95° C. and 1minute at 68° C. was repeated 25 times, followed by a 10-minuteelongation reaction at 68° C. Following agarose gel electrophoresis, theamplified DNA fragment was collected from the gel, digested withrestriction enzymes BamH I and Xho I, cloned to the BamH I and Xho Isites of protein expression vector pScc Tag2 B (Invitrogen), andintroduced into E. coli DH5α to obtain E. coli DH5α/Human MAY I-pSecTag2 B. After the nucleotide sequence of the inserted cDNA was confirmedas in Example 7, a cDNA sequence (SEQ ID No. 15) encoding the protein(human MAY-I) from amino acid 661 through the C-terminal amino acid ofhuman IHRP was obtained.

EXAMPLE 12 Cloning of DNA Encoding Human MAY-I (2)

A transformant containing cDNA homologous to DNA encoding rat MAY-I wascloned from a human liver cDNA library (Clontech), using DNA encodingrat MAY-I as the probe. Analysis of the nucleotide sequence of this cDNAfragment confirmed that the cDNA fragment included the coding regionsbetween base 1424 and the C-terminal of the human IHRP cDNA sequence(SEQ ID No. 16).

EXAMPLE 13 Preparation of Protein (Human MAY-I) Obtained in Example 11Corresponding to Amino Acids Between 661 and the C-Terminal of HumanIHRP, and Immunosuppressive Activity of Prepared Recombinant Human MAY-I

The Human MAY I-pSec Tag2 B prepared in Example 11 was transfected intoCOS 7 cells by the DEAE Dextran method. The day after transfection itwas transferred to FBS-free medium and cultured for 24 hours, afterwhich the culture supernatant was collected and recombinant Human May-Itherein isolated and purified according to the methods shown in Example9. This Human MAY-I exhibited concentration-dependent immunosuppressiveactivity in human MLR (FIG. 11).

Industrial Applicability

Since the protein of the present invention has immunosuppressiveactivity, it is useful as a prophylactic or therapeutic agent forauto-immune disorders (rheumatism, psoriasis and the like) or allergicdisorders (allergic asthma (bronchial asthma), allergic rhinitis,allergic conjunctivitis, allergic dermatitis (atopic dermatitis),pollinosis and the like), and antirejection drug.

1. A protein (a) or (b) below: (a) a protein comprising the amino acidsequence shown by SEQ ID No. 14; (b) a protein comprising the amino acidsequence as defined in (a) wherein one or more amino acids are deleted,replaced or added and having immunosuppressive activity.
 2. A protein(a) or (b) below: (a) a protein having the amino acid sequence shown bySEQ ID No. 1; (b) a protein comprising the amino acid sequence asdefined in (a) wherein one or more amino acids are deleted, replaced oradded and having immunosuppressive activity.
 3. A protein (a) or (b)below: (a) a protein comprising the amino acid sequence shown by SEQ IDNo. 8; (b) a protein comprising the amino acid sequence as defined in(a) wherein one or more amino acids are deleted, replaced or and addedand having immunosuppressive activity.
 4. A gene encoding the protein asdefined in any one of claims 1 through
 3. 5. A gene comprising a DNA (a)or (b) below: (a) a DNA comprising the nucleotide sequence shown by SEQID No. 15; (b) a DNA which hybridizes under stringent conditions with aDNA comprising the nucleotide sequence as defined in (a), and whichencodes a protein having immunosuppressive activity.
 6. A genecomprising a DNA (a) or (b) below: (a) a DNA comprising the nucleotidesequence shown by SEQ ID No. 2; (b) a DNA which hybridizes understringent conditions with a DNA comprising the nucleotide sequence asdefined in (a), and which encodes a protein having immunosuppressiveactivity.
 7. A gene comprising a DNA (a) or (b) below: (a) a DNAcomprising the nucleotide sequence shown by SEQ ID No. 9; (b) a DNAwhich hybridizes under stringent conditions with DNA comprising thenucleotide sequence as defined in (a), and which encodes a proteinhaving immunosuppressive activity.
 8. A protein having an amino acidsequence encoded by the gene as defined in any one of claims 4 through7.
 9. A vector containing the gene as defined in any one of claims 4through
 7. 10. A transformant containing the vector as defined in claim9.
 11. A method of manufacturing a protein comprising: Step 1 ofculturing the transformant as described in claim 10; and Step 2 ofcollecting a protein having immunosuppressive activity from the cultureobtained in said step.
 12. A medicinal composition containing any one ofthe protein defined in any one of claims 1 through 3 or claim 8 as anactive ingredient, together with a pharmacologically acceptable carrier.13. The medicinal composition as defined in claim 12, wherein themedicinal composition is an immunosuppressant.
 14. The medicinalcomposition as defined in claim 13, wherein the immunosuppressant is aprophylactic or therapeutic agent for an auto-immune disorder orallergic disorder, or an antirejection drug.
 15. The medicinalcomposition as defined in claim 14, wherein the auto-immune disorder isrheumatism or psoriasis.
 16. The medicinal composition as defined inclaim 14, wherein the allergic disorder is bronchial asthma, allergicrhinitis, allergic dermatitis or pollinosis.